Localization by segmental deletion analysis and functional characterization of a third actin-binding site in domain 5 of scinderin.
Scinderin is a Ca2+-dependent actin filament severing protein present in a variety of secretory cells. Previous work suggests that scinderin-evoked cortical F-actin disassembly is required for secretion because local disassembly of cortical cytoskeleton allows secretory vesicle exocytosis (Vitale, M. L., Rodríguez Del Castillo, A., Tchakarov, L., and Trifaró, J.-M. (1991) J. Cell Biol. 113, 1057-1067). Scinderin has six domains each containing three internal sequence motifs, two actin, and two phosphatidylinositol disphosphate-binding sites in domains 1 and 2. In this paper we report the presence of another actin-binding site at the NH2-terminal of domain 5 (Sc511-518). This site binds actin in a Ca2+-independent manner and a recombinant fragment (Sc5-6 or Sc502-715) containing this site binds to actin-DNase-I-Sepharose 4B beads, co-sediments with actin and is able to nucleate actin assembly. Recombinant ScL5-6, a fusion protein devoid of the actin-binding site (Sc519-715), did not exhibit these properties. Moreover, Sc-ABP3, a peptide constructed with sequence (RLFQVRRNLASIT) identical to Sc511-523 blocked the binding of Sc5-6 to actin. Sc5-6 and Sc-ABP3 also prevented the actin severing activity of recombinant full-length scinderin (r-Sc) and inhibited the potentiation by r-Sc of Ca2+-evoked release of serotonin from permeabilized platelets. On the other hand, ScL5-6 failed to block the effect of r-Sc on platelet serotonin release. Sc1-4,6, a construct devoid of domain 5, was able to sever but unable to nucleate actin, indicating that an actin nucleation site of scinderin was in domain 5. The results suggest that scinderin, in addition to binding actin on sites present in domains 1 and 2, must bind actin on a third site in domain 5 to sever and nucleate actin effectively.
Marcu
M G
MG
Secretory Process Research Program, Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
Zhang
L
L
Elzagallaai
A
A
Trifaró
J M
JM
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D018260:Gelsolin; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion
D000199:Actins; D018260:Gelsolin; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C065395:scinderin